Fig. 5.

Fig. 5.

Using PCR tagging for cellular expression analysis reveals vamp cellular diversity. (A) Cellular expression tagging strategy in which a membrane-bound fluorescent protein sequence (memFP) followed by a ‘self-cleaving’ P2A peptide is knocked into the N terminus of a gene of interest. During mRNA translation, ribosomal skipping leads to separate production of memFP outlining the morphology of the expressing cells and of intact endogenous protein. HA, homology arms; m7G/AAAA, mRNA 5′-cap and polyadenylation signal. (B) Efficiency of cellular expression tagging of vamp1/2/3 homologues. Bars represent one injection round; the number of 3-5 dpf larvae analyzed n is indicated within bars. Error bars indicate 95% confidence intervals for FP⁺ expression (Wilson/Brown calculation). Modified HDR template concentration indicated below bars; 30 µM IDT HDR enhancer v2 or 80 µM L755507 was added in vamp1/2 or in vamp3 tagging injections, respectively. (C) vamp2 cellular expression tagging shows extensive neuronal expression throughout the spinal cord, e.g. in sensory Rohon–Beard neurons, CSF-contacting neurons and motor neurons. (D) vamp1 cellular expression tagging reveals expression in motor neurons, premotor reticulospinal neurons (e.g. the Mauthner cell) and interneuron subtypes such as commissural primary ascending (COPA) neurons. Brackets indicate region of the single z-slice shown above. (E) vamp3 cellular expression tagging reveals primarily glial expression e.g. in myelinating oligodendrocytes, radial glia and astrocyte-like cells in the ventral spinal cord. Brackets indicate region of z-slice shown below. Arrowheads point to examples of the labelled cells or structures in each panel. MIP, maximum intensity projection of z-stack. Scale bars: 5 µm.